Genomic and functional portrait of multidrug-resistant, hydrogen sulfide (H2S)-producing variants of Escherichia coli

Atypical Escherichia coli forms exhibit unusual characteristics compared to typical strains. The H2S-producing variants of some atypical E. coli strains cause a wide range of illnesses in humans and animals. However, there are sparse reports on such strains worldwide. We performed whole-genome sequencing (WGS) and detailed characterization of four H2S-producing E. coli variants from poultry and human clinical sources in Dhaka, Bangladesh. All four isolates were confirmed as E. coli using biochemical tests and genomic analysis, and were multidrug-resistant (MDR). WGS analysis including an additional Chinese strain, revealed diverse STs among the five H2S-producing E. coli genomes, with clonal complex ST10 being detected in 2 out of 5 genomes. The predominant phylogroup detected was group A (n = 4/5). The blaTEM1B (n = 5/5) was the most predominant extended-spectrum beta-lactamase (ESBL) gene, followed by different alleles of blaCTX-M (blaCTX-M -55,-65,-123; n = 3/5). Multiple plasmid replicons were detected, with IncX being the most common. One E. coli strain was classified as enteropathogenic E. coli. The genomes of all five isolates harbored five primary and four secondary function genes related to H2S production. These findings suggest the potential of these isolates to cause disease and spread antibiotic resistance. Therefore, such atypical E. coli forms should be included in differential diagnosis to understand the pathogenicity, antimicrobial resistance and evolution of H2S-producing E. coli.


Introduction
Escherichia coli is one of the most genetically diverse and versatile organisms, varying from commensal/avirulent to highly specialized pathogenic strains. E. coli can thrive in several niches, including hosts and in the environment (Kaper et al., 2004;Braz et al., 2020). The variant strains of E. coli may act as facultative or obligate pathogens (Köhler and Dobrindt, 2011). The facultative strains of pathogenic E. coli survive in the intestinal tract and often cause opportunistic infections when reaching suitable extraintestinal sites (Nataro and Kaper, 1998;Kaper et al., 2004). In contrast, enteric obligate pathogens can cause infections in different conditions that range from moderate to severe diarrhea, and can sometimes cause lethal gastrointestinal infections (Nataro and Kaper, 1998).
Pathogenic variants of E. coli are responsible for infections in a variety of animals, more commonly in humans and poultry (Bélanger et al., 2011;Hussain et al., 2017). Pathogenic E. coli has been reported in livestock, including poultry, cattle, and swine (Bélanger et al., 2011). Animal reservoirs of pathogenic E. coli are responsible for diseases in animals, but can spread the infections to humans, including antimicrobial resistant (AMR) strains (Bélanger et al., 2011). Traditionally, biochemical tests have been used for differentiating and identifying members of Enterobacteriaceae, including hydrogen sulfide (H 2 S) gas (Zabransky et al., 1969). H 2 S can be synthesized by bacteria such as Campylobacter, Salmonella, Citrobacter, and Erwardsiella and Proteus species on TSI or KIA media (Blachier et al., 2019). This distinct characteristic feature of H 2 S production by certain bacteria within Enterobcateriacea is used as a bacterial identification test in diagnostic microbiology. E. coli generally does not produce H 2 S, which differentiates it from the other members of Enterobacteriaceae (Percival et al., 2004). However, a few studies worldwide have reported the presence of atypical H 2 Sproducing E. coli forms in humans and animals (Darland and Davis, 1974;Maker and Washington, 1974;Magalhães and Vance, 1978). They have been isolated from poultry, swine and clinical human urine specimens.
The enzyme 3-mercaptopyruvate sulfurtransferase (3MST) is reported to be mainly responsible for the synthesis of endogenous H 2 S in Enterbacteriaceae (Mironov et al., 2017). Some studies have also demonstrated the transmissibility of H 2 S-producing traits between strains via plasmids (Jones and Silver, 1978;Magalhães and Vance, 1978). Although the physiological function of endogenously produced hydrogen sulphide is not clearly defined, recent studies have pointed out its role in protecting bacteria against antibiotics and host defence systems (Mironov et al., 2017;Rahman et al., 2020). A plausible explanation for this is that the antibiotics induce oxidative stress in bacteria by increasing the levels of reactive oxygen species; in response to this, the bacteria produces H 2 S which in turn stimulates enzymes such as superoxide dismutase (SOD) and catalase that alleviates the effect of reactive oxygen species, and thereby reduces the efficacy of antibiotics contributing to AMR (Eswarappaápradeep, 2017). Also, studies have demonstrated the role of bacterial H 2 S production in defence against host immunity by making them resistant to leukocytes-mediated killing via unknown mechanisms (Toliver-Kinsky et al., 2019;Rahman et al., 2020).
The accurate identification of H₂S-producing variants of E. coli in diagnostic laboratories is an important step for initiating effective infection management. There is a need to raise awareness of this unusual type of E. coli form that occurs frequently but differs in its inability to produce H 2 S compared to the typical E. coli forms. Therefore, this study aimed to perform bacteriological, biochemical and genomic characterization of H 2 S-producing variants of E. coli from healthy poultry and human clinical sources in Dhaka, Bangladesh. We present the first report on the genomic characterization of H 2 S-producing variants of E. coli from Bangladesh and that from South Asia.

Ethics statement
The study protocols were approved by the Research Review committee and Ethics Review Committee of icddr,b, Dhaka, Bangladesh (PR-23045).

Bacterial strains
A surveillance study was conducted between 2019 and 2021 to investigate the genomic-based epidemiology of AMR Enterobacteriaceae in healthy poultry and human clinical samples in Dhaka, Bangladesh (Mazumder et al., 2020a(Mazumder et al., , 2021(Mazumder et al., , 2022. During that study, we detected four lactose fermenting E. coli colonies but with an atypical biochemical feature of H 2 S production. These were confirmed to be E. coli by the methods described hereafter. Three (BD7, BD8, BD9) of these H 2 Sproducing E. coli originated from raw poultry meat and one isolate (BD_CL10) was cultured from a urine sample of a suspected urinary tract infection patient in Dhaka, Bangladesh. Thus, from a collection of 96 poultry E. coli isolates and 204 human clinical E. coli study isolates, we could obtain three and one H 2 S-producing E. coli isolates, respectively. These four H 2 S-producing E. coli isolates then formed the basis of this study, and underwent various tests and whole genome sequencing (WGS). One H 2 S positive E. coli genome from China (Biswas et al., 2020) was used for the in-silico analysis together with the four studied H 2 S positive E. coli genomes sequenced in this study.

Biochemical characterization and antimicrobial susceptibility
The complete bacteriological and biochemical characteristics of H 2 S-producing variants of E. coli strains are summarized (Table 1). The biochemical identification included the following tests; kligler iron agar (KIA) test, motility, indole and urease (MIU) test, citrate and acetate utilization test, catalase test, oxidase test, vogas-proskauer test, gelatin liquefaction and ONPG tests. In addition, colonies were plated on Muller-Hinton agar containing 0.68% of sodium thiosulfate plus 0.08% of ferric ammonium sulfate as previously described (Park et al., 2015). The isolates that mimic E. coli in all aspects except H 2 Sproduction in Kligler iron agar (KIA) and Muller-Hinton agar (with sodium thiosulfate and ferric ammonium sulfate) were carried forward in this study. These preliminary identified 4 H 2 S-positive E. coli isolates were subjected to additional tests, including fermentation of sugars and decarboxylation reaction of amino acids (Mazumder et al., 2022). Further, the possibility of Salmonella spp. was ruled out by slide agglutination test using O, O1 polyvalent and VI Salmonella antisera (Denka Seiken Co. Ltd. Tokyo, Japan). The API 20E kit (bioMérieux) was used to generate the analytical profile index (Table 1). Haemolysis was evaluated using 5% sheep blood agar plates. Disk diffusion method was employed to determine the antimicrobial susceptibility. The Clinical and Laboratory Standards Institute (CLSI) guidelines (Weinstein, 2019) were followed. Twenty commercially available antibiotic disks (Oxoid, US) covering 11 antimicrobial classes were tested (see Table 2). The intermediate susceptibility was described as non-susceptible. Isolates were termed multi-drug resistant (MDR) Frontiers in Microbiology 03 frontiersin.org if refractory to at least one antibiotic from three or more antimicrobial classes (Magiorakos et al., 2012).

Whole-genome sequencing
Total bacterial DNA was extracted using the Maxwell RSC Instrument and Culture Cell DNA extraction Kit (Promega) for gramnegative bacteria with an additional RNaseA treatment (Baddam et al., 2020;Mazumder et al., 2020b,c). The DNA QC was assessed by Nanodrop spectrophotometer (Thermo Fisher Scientific, US), Quantus Fluorometer (Promega, US) and by 1% agarose gel electrophoresis. The paired-end bacterial WGS libraries were constructed from 220 to 250 ng of genomic DNA using the Illumina DNA Prep kit as per the manufacturer's instructions (Mazumder et al., 2021). The pooled libraries thus obtained were sequenced at the icddr,b Genome Centre on Illumina NextSeq 500 system to obtain 100-to 150-fold coverage for each genome using a NextSeq v2.5 Mid Output reagent kit (2 × 150 bp) (Mazumder et al., 2022;Monir et al., 2023).

In silico sequence analysis
The reads of H 2 S-producing E. coli were uploaded to the KmerFinder v3.2 Larsen et al., 2014) for species confirmation. The phylogenetic groups were ascertained using Clermon Typing tool (Beghain et al., 2018). The sequence types (STs), clonal complex and pathovars were predicted employing the Achtman7 seven-locus scheme at EnteroBase v1.
Single nucleotide polymorphism-based core genome phylogeny We used Snippy (v4.4.0) software (Seemann, 2015) with default parameters to obtain the reference-guided multi-fasta consensus alignment of 5 H 2 S-producing E. coli genomes using E. coli MG1655 as the reference. Gubbins software (v3.2 5) (Croucher et al., 2015) was used to filter true point mutations from those arising from recombination. The phylogenetic tree was determined using RaxML (v8.2.12), utilizing the Generalized Time Reversible substitution model and a GAMMA distribution to account for rate heterogeneity (Stamatakis, 2014). Finally, the phylogenetic tree was displayed using IToL (Letunic and Bork, 2016).

Bacterial characteristics
Four H 2 S-positive E. coli variants were identified that formed a black precipitate after overnight incubation in an aerobic environment in the Kligler iron agar (KIA) and Mueller Hinton agar medium enriched with both sodium thiosulfate and ferric ammonium sulfate (Figure 1). Attempts to agglutinate the strains with polyvalent Salmonella antisera yielded negative results. When streaked on CHROMagar Orientation media, E. coli produced small, pink-red colonies that were characteristic of the species. Routine biochemical tests identified the strains as E. coli, except for their ability to reduce thiosulfate to H 2 S (Table 1). All four isolates were gram-negative rods, motile, oxidase-negative, catalase-positive and indole positive. All isolates tested showed a lack of urease and Voges-Proskauer reaction, and they did not grow on the Simmons Citrate agar medium. Nonetheless, all isolates exhibited a positive result for O-nitrophenyl-beta-Dgalactopyranoside (ONPG) and carried out fermentation of glucose and lactose sugars, leading to gas production ( Figure 1). The API results revealed two distinct profiles, 5,544,512 (n = 3), and 5,544,552 (n = 1) and confirmed isolates as E. coli with 99% certainty (Table 1). The optimum growth temperate ranged between 26° to 42°C and they produced gamma-haemolysis on sheep blood agar.

Molecular and phylogenomic analysis of H 2 S-positive E. coli genomes
This analysis included the four in-house strains and a genome of H 2 S-producing E. coli reported from China (China_H 2 S). WGS-based species identification confirmed all the isolates as E. coli. Across the five H 2 S-producing E. coli strains, the average genome size was 4,853,304 bp (range 4,501,832 to 5,198,676) with an average GC content of 50.6% (range: 50.4 to 50.9%). The genome assemblies had an average coverage of 138-fold, with a range of 102 to 206-fold (Table 3). They had five distinct STs, which comprised ST10, ST48, ST12434, ST189, and ST12066. We detected four clonal complexes that included CC10 (two strains from human sources) followed by CC155, CC165 and CC206, representing one strain each (Figure 2). We identified four isolates (80%) belonging to commensal phylogroup A and one isolate (20%) to B1 phylogroup. All H 2 S-producing E. coli isolates exhibited distinct serotypes and CH types. A phylogenetic tree was constructed for five H2S-producing E. coli genomes using the MG1655 genome as a reference, by aligning the core genome single nucleotide polymorphisms (SNPs). The studied H 2 S-producing E. coli strains were found to be relatively diverse. However, the three poultry H 2 Spositive strains from Bangladesh clustered together, with human strains adjacent to this cluster. The molecular characteristics did not correlate with the source of origin or the phylogenetic clustering of the H 2 S-producing E. coli isolates (Figure 2). All five H 2 S-producing E. coli genomes harbored five primary hydrogen sulfide-producing genes; cysteine aminotransferase (aspC), cysteine desulfhydrase (dcyD), 3-mercapto pyruvate sulfurtransferase (sseA), yhaOM operon (yhaM, yhaO). Whereas the methionine gamma-lyase (mgl) gene was completely absent. Two of the four secondary function genes, cysteine synthase A (cysK) and cystathionine beta-lyase (metC) were found in all four strains. However, the other genes such as cysteine synthase B (cysM), and cystathionine beta-lyase-like repressor of maltose regulon (malY) are sparingly present in poultry isolates. The erroneous H 2 S-producing genes, including cysteine desulfurase (iscS) and tryptophanase (tnaA) were observed in all isolates. As expected, all three class of cysteinedegradation genes were found on chromosomes (Figure 2; Table 4).

Plasmid replicon types
PlasmidFinder identified 20 unique plasmid replicon groups (Table 5). All five isolates harbored multiple plasmid replicons. The plasmid replicons identified include IncFII (pHN7A8), IncFII Phylogenetic relationships among sequenced Hydrogen Sulfide (H 2 S)-Producing Escherichia coli genomes. The maximum likelihood phylogenetic tree is based on the alignment of detected core genomes with the MG1655 genome strain as a reference genome. The country of origin, isolation date, sample source, phylogroups, pathovar, MDR status, STs, clonal complex, serogroups, genes responsible for hydrogen sulfide production via cysteinedegradation and CH types are shown next to the tree.

Prophage analysis
We detected intact prophages in the Bangladeshi H 2 S-producing E. coli strains, but not in the Chinese isolate. The poultry strains harbored two to three intact prophage sequences, while the human clinical strains carried four intact prophage sequences. Incomplete prophage sequences ranged from four to seven in poultry strains and one in human strains, while questionable prophage sequences ranged from zero to two in poultry strains and were absent in human strains identified in Bangladesh. However, five incomplete prophage sequences were detected in H 2 S-producing E. coli from China. The most common phages in the H 2 S-producing E. coli strains were Klebsiella phage 4 LV-2017 (2/5) and Shigella phage SfII (2/5), with both phages present together in a single H 2 S-producing human clinical E. coli strain (Table 3).

CRISPR-CAS system
The CRISPR-CAS system subtype I-A and I-E were found to be the most prevalent in the five H 2 S-producing E. coli genomes. All H 2 S-producing E. coli strains obtained from both poultry and human sources had only one CRISPR locus. The number, nucleotide sequence, and average length of repeats and spacers were similar in all H 2 Sproducing E. coli strains, but they varied in the quantity of repeats and spacer units. The human clinical strain BDCl_10 was comparable to poultry strains, except that it had a higher number of repeats and spacers than the poultry strains (as shown in Table 3).

Antimicrobial resistance phenotypes and genotypes
All four (100%) H 2 S-producing E. coli isolates from Bangladesh were resistant to ampicillin, nalidixic acid, ciprofloxacin, sulfamethoxazoletrimethoprim, doxycycline, and tigecycline. While they were all sensitive to amikacin, imipenem, meropenem, and colistin. However, three isolates (75%) were resistant to ceftazidime, cefotaxime, cefepime, cefuroxime, and ceftriaxone. Whereas 50% of isolates were resistant to chloramphenicol, fosfomycin, and gentamicin. All four H 2 S-producing E. coli isolates were classified as MDR.

Discussion
Production of hydrogen sulphide (H 2 S) is seen in many members of Enterobacteriaceae. However, it is well established that E. coli strains are H 2 S non-producers. H 2 S non-production is one of the key characteristics used to identify E. coli in laboratory tests. Nonetheless, a fraction of H 2 S-producing E. coli variants has previously been identified in animal and human infections (Maker and Washington, 1974;Sogaard, 1975;Magalhães and Vance, 1978). By studying H 2 S-producing E. coli, researchers can better understand the biology and behavior of such variants and develop improved diagnostic tests. Further, a comprehensive characterization of H 2 S-producing E. coli including analysis of genomic features was needed. To address this, we conducted a thorough investigation of four H 2 S-producing E. coli variants by utilizing whole-genome sequencing (WGS) in combination with comprehensive microbiological and biochemical testing.
The four bacterial isolates were recovered from poultry and human clinical samples in Dhaka, Bangladesh, as part of a larger surveillance study. These isolates biochemically mimic typical E. coli for all reactions except for one reaction, the H 2 S production. The prevalence of H 2 S-producing variants in our study can be estimated at 3% (3/96) in poultry and 0.5% (1/204) in clinical E. coli isolates. However, this may not reflect the true prevalence figures, as in this study, the primary specimens were not screened targeting H 2 Sproducing E. coli. But only the archived E. coli isolates were tested. However, our estimates of prevalence are similar to those previously reported (Maker and Washington, 1974;Sogaard, 1975;Weber et al., 1981). Our and other reports reveal that H 2 S-positive strains of E. coli are not uncommon among poultry and human clinical samples (Braunstein and Mladineo, 1974;Maker and Washington, 1974;Sogaard, 1975;Traub and Kleber, 1975;Magalhães and Vance, 1978;Weber et al., 1981;Barbour et al., 1985). Many such variants are probably misidentified in laboratories, such as Citrobacter, Arizona and Salmonella (Darland and Davis, 1974). This misidentification stems from the production of black precipitate on KIA or TSI medium. It is also possible that acid production sometimes masks H 2 S production due to lactose fermentation (Magalhães and Vance, 1978). Muller-Hinton agar supplemented with sodium thiosulfate and ferric ammonium sulfate media is considered superior to KIA agar media for identifying H 2 S production. The utility of the same has been demonstrated in this study. However, the CHROMagar Orientation media could not differentiate between typical E. coli and H 2 Sproducing E. coli variants. Primary screening with this media can effectively screen typical E. coli and H 2 S-producing E. coli variants in a single step.
The studied H 2 S-producing E. coli strains mainly belonged to the commensal phylogenetic groups A (80%,4/5) and B1 (20%, 1/5). Several reports confirm that phylogroups A and B1 were the most prevalent among E. coli isolates, particularly in the gut microbiome (Li et al., 2010;Stoppe et al., 2017). The H 2 S-producing E. coli strains of human origin, isolated from Bangladesh and China, belonged to the worldwide predominant clonal complex CC10. CC10 group of strains belong to emerging clone of extra-intestinal pathogenic E. coli (ExPEC) (Manges et al., 2019). They are isolated from a wide range of niches including clinical settings, food animals and environment (Manges et al., 2019). They are also known to be associated with wide range of AMR and virulence genotypes (Massella et al., 2021). This group of E. coli needs close monitoring to safeguard public health (Hussain et al., 2023). We identified 8-13 prophage regions in H 2 Sproducing E. coli, of which 2-4 were found intact. Klebsiella phage 4 LV-2017 and Shigella phage SfII were the predominant bacteriophages detected. The existence of a higher number of phage elements (8 to 13) in poultry strains compared to the human clinical strain (5) may indicate more horizontal gene transfer (HGT) events that brought in more toxin genes in poultry strains than in the human clinical strain. The CRISPR-Cas system confers immunity against viruses and plasmids (Horvath and Barrangou, 2010). Investigation of the CRISPR-Cas system in H 2 S-producing E. coli strains indicated that it Heat map demonstrating the antimicrobial resistance (AMR) genes profile of 5 Hydrogen Sulfide (H 2 S)-producing Escherichia coli genomes. Dark black blocks represent the presence of AMR genes, and light green blocks represent the absence of particular genes. Microbiology  11 frontiersin.org was conserved in both poultry and human clinical H 2 S-producing E. coli isolates. Previous work has identified cysteine-degradation genes in H 2 Sproducing bacteria and classified them into primary, secondary and erroneous categories based on their functions (Braccia et al., 2021). Most primary producer genes (aspC, dcyD, sseA, yhaOM operon) were present in all H 2 S-producing E. coli strains. In the case of secondary producer genes, we observed inconsistent results. But all erroneous genes were present in the study isolates. We found all genes related to H 2 S production on chromosomes, which is in line with the previous report (Braccia et al., 2021).

Frontiers in
The patterns of antibiotic resistance were similar for human and poultry isolates. High resistance rates were observed for ampicillin, ciprofloxacin, nalidixic acid, trimethoprim and sulfamethoxazole, doxycycline and cephalosporin. Our findings show partial agreement with the previous report on H 2 S-producing E. coli (Braunstein and Mladineo, 1974;Maker and Washington, 1974;Sogaard, 1975;Traub and Kleber, 1975;Magalhães and Vance, 1978;Weber et al., 1981;Barbour et al., 1985;Park et al., 2015). The H 2 S-producing E. coli isolates contained multiple plasmids. The major replicon types were IncX (4/5; 80%) and IncF (3/5; 60%). As per earlier reports, these plasmid replicons were associated with fluoroquinolone resistance and bla CTX-M-group in humans and livestock E. coli (Phan et al., 2015;Sun et al., 2017). As healthy animals and humans were found to harbor H 2 S-producing E. coli (Sogaard, 1975;Biswas et al., 2020), the presence of these plasmids may contribute as careers of antibiotic resistance in microbiomes. The results of our study suggests that aminoglycosides and carbapenem antibiotics are effective candidates against these strains. However, this cannot be generalized due to several limitations of our study and it is always better to initiate evidence-based treatment of diseases arising from infectious agents.
All isolates were predicted as human pathogens as per their pathogenicity score determined by in silico analysis. The studied H 2 Sproducing E. coli isolates harbored at least 30 virulence factors. Among them, poultry isolates had more virulence genes (40-57 VFs) than human samples. The H 2 S-producing E. coli isolates harbored a wide range of virulence factors encoding E. coli laminin-binding fimbriae (ELF) (elfA,C,D,G), Hemorrhagic E. coli pilus (HCP) (hcpA-B), Type I fimbriae (fimD, fimF, fimG, fimH) and Non-LEE encoded TTSS effectors (espL1, espL4, espR1, espX4). The intimin (eae) gene, a marker for enteropathogenic E. coli, was observed in one H 2 Sproducing E. coli isolate (BD8) belonging to ST189. This indicates that E. coli pathotypes also exhibit H 2 S production features or vice versa. Therefore, virulence genes play an important role in the pathogenicity of H 2 S-producing E. coli strains. Also, the convergence of wide range of AMR and virulence genotypes is a cause of great concern (Massella et al., 2021). These observations warrant studying the role of H 2 Sproducing E. coli isolates in different infections for developing effective treatments and preventive measures.
In conclusion, this study investigated H 2 S-producing E. coli variants recovered from poultry and human clinical samples in Dhaka, Bangladesh. The isolates were confirmed as E. coli by routine biochemical tests and WGS-based species identification. The H 2 Sproducing isolates exhibited relatively diverse molecular characteristics with no correlation between the source of origin or the phylogenetic clustering of the isolates. The study also found high rates of AMR and extensive virulence gene repertoire in these isolates. The findings of this study highlight that the genomic features, antibiotic resistance and virulence potential of H 2 S-producing E. coli resemble the typical E. coli forms. Therefore, we suggest the need for continued surveillance and genomic characterization of atypical E. coli forms like H 2 S-producing E. coli to better understand the characteristics of such variants and improve diagnostics and treatment outcomes.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/ genbank/, PRJNA882002, PRJNA714244.